Investigating phenotypic changes in wound biofilms in response to antimicrobial treatment using Raman Spectroscopy.

使用拉曼光谱研究伤口生物膜对抗菌治疗的表型变化。

• 批准号: 2902109
• 金额: --
• 依托单位: University of Strathclyde
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2902109
• 关键词: Investigating; phenotypic; changes; wound; biofilms

Wound infections such as diabetic foot ulcers (DFUs) can be highly problematic in terms of clinical management. Such infections carry a significant socioeconomic burden, costing the NHS an estimated £8.3b in 2017/18. DFUs have significant mortality rates, with 5-year mortality rates comparable with cancer. However, worryingly this is not reflected in the level of funding for chronic wound compared to cancer research.The main difficulty that clinicians face during the treatment of chronic wounds is the risk of infection at the wound site, arising from the accumulation of microbial aggregates in the wound bed. Infected wounds arise from the formation of complex biofilms that can be largely resistant to antibiotic therapy: most of which are polymicrobial in nature . This further complicates treatment regimes to alleviate specific pathogenic microorganisms within the mixed community. Given the increased complexity of the wound microbiome as well as the increasing threat of antimicrobial resistance, clinicians are faced with the challenging prospect of targeted topical therapy or systemic antibiotics. This can be extremely difficult as point-of-care technologies in wound care are limited with regards to microbial detection, with clinicians relying on basic culture or molecular methodologies that can take days for results. Thus there remains a requirement for rapid, sensitive, yet non-invasive methodologies for microbial identification at the bedside, to aid clinical therapy choices during wound management.Raman is an information rich vibration technique that gives label-free biochemical information at the sub-cellular level, however high-resolution imaging at a single cell level is a relatively slow process making it challenging for live cell imaging. Stimulated Raman scattering (SRS) is a more sensitive and much faster technique which can provide high resolution information on larger cell populations compared with normal Raman. SRS provides detailed biochemical information with sub-cellular resolution including determination of protein, lipids and nucleic acid location. Due to its speed and ability to image larger areas in a label free manner, it has great potential for imaging bacterial biofilms giving both biochemical information as well as structural information on biofilm formation, as well as changes to biofilm in response to antimicrobial treatments. To date there is evidence that different bacterial species exhibit unique spectral profiles depending on their chemical structures at a cellular level. Existing research is largely limited to single cells, with few studies investigating biofilm kinetics using SRS.The overall aims of the proposal are to test the feasibility of using SRS to monitor biofilm formation and assess unique Raman spectral profiles or signatures of different bacterial and fungal species related to wound infections.1 Monitoring growth dynamics of mono-species wound biofilm models using SRS - comparing spectral profiles in different clinically-relevant microbial species (gram+, gram- bacterial and fungal organisms) during biofilm attachment, maturation and dispersal.2 Investigating mono-species biofilm response to antibiotic therapy using a variety of microbiological, molecular, microscopic and spectroscopic techniques - SRS to be used to assess chemical signatures in the biofilms in response to bacterio- (or fungi-) static or -cidal antibiotics and other novel compounds.3 Assessing changes in biofilm formation in a multi-species biofilm model using SRS. Visualizing phenotypic changes in biofilm chemical structures containing a consortia of different microbial species.4 SRS to be used to profile real-world biofilms (those generated from clinical samples from wound infections which will be generated through an existing EPSRC grant EP/V005839/1). Characteristic changes in chemical structures identified by Raman Spectroscopy using simple biofilm models with known composition.

伤口感染，如糖尿病足溃疡（DFU），在临床管理方面可能存在很大问题。这种感染带来了巨大的社会经济负担，NHS在2017/18年度估计花费了83亿英镑。DFU有显著的死亡率，5年死亡率与癌症相当。然而，令人担忧的是，与癌症研究相比，这并没有反映在慢性伤口的资金水平上。临床医生在慢性伤口治疗过程中面临的主要困难是伤口部位感染的风险，这是由伤口床中微生物聚集体的积累引起的。感染的伤口是由复杂的生物膜的形成引起的，这些生物膜在很大程度上对抗生素治疗具有抗性：其中大多数在本质上是多微生物的。这进一步使减轻混合群落内特定病原微生物的处理方案复杂化。鉴于伤口微生物组的复杂性增加以及抗菌素耐药性的威胁增加，临床医生面临着靶向局部治疗或全身抗生素的挑战性前景。这可能是非常困难的，因为伤口护理中的即时技术在微生物检测方面受到限制，临床医生依赖于基础培养或分子方法，可能需要几天的时间才能获得结果。因此，仍然需要快速、灵敏、但非侵入性的方法来在床边进行微生物鉴定，以在伤口处理期间辅助临床治疗选择。拉曼是一种信息丰富的振动技术，其在亚细胞水平上提供无标记的生化信息，然而，在单细胞水平上的高分辨率成像是一个相对缓慢的过程，这使得活细胞成像具有挑战性。受激拉曼散射（SRS）是一种比普通拉曼更灵敏、更快速的技术，它可以提供更大细胞群体的高分辨率信息。SRS提供了详细的生化信息与亚细胞分辨率，包括确定蛋白质，脂质和核酸的位置。由于其速度和以无标记方式成像较大区域的能力，它具有成像细菌生物膜的巨大潜力，提供生物膜形成的生化信息和结构信息，以及响应于抗微生物处理的生物膜变化。迄今为止，有证据表明，不同的细菌物种表现出独特的光谱分布，这取决于它们在细胞水平上的化学结构。现有的研究主要限于单细胞，该提案的总体目标是测试使用SRS监测生物膜形成的可行性，并评估与伤口感染相关的不同细菌和真菌物种的独特拉曼光谱分布或特征。1使用SRS监测单物种伤口生物膜模型的生长动力学。比较不同临床相关微生物物种的光谱分布（革兰氏+，革兰氏-细菌和真菌生物体）在生物膜附着，成熟和分散过程中。2研究单种生物膜对抗生素治疗的反应，使用各种微生物，分子，显微镜和光谱技术- SRS用于评估生物膜中响应细菌（或真菌）静态或杀菌抗生素和其他新型化合物的化学特征。3使用SRS评估多物种生物膜模型中生物膜形成的变化。可视化生物膜化学结构中包含不同微生物物种的聚生体的表型变化。4 SRS用于描述真实世界生物膜（由伤口感染的临床样本生成的生物膜，将通过现有EPSRC授权EP/V005839/1生成）。通过拉曼光谱法使用具有已知组成的简单生物膜模型识别化学结构的特征变化。